Objective  To obtain high-yield and high-purity recombinant Irisin protein by recombinant expression and purification in Escherichia coli, and to lay a foundation for the subsequent study of Irisin function.

Methods  The CDS sequence of the Irisin gene was codon optimized for gene synthesis, and the recombinant plasmid pET-30a-Irisin was constructed by a one-step cloning and ligation method, and was transformed into Escherichia coli expression receptor cells Rosetta (DE3). The expression was induced by IPTG at a final concentration of 0.5 mM for 30 h at 15 °C and 100 rpm. The bacteria were collected at the end of induced expression, and the bacterial cells were broken by ultrasonic waves, the supernatant was obtained by centrifugation and purified by Ni-IDA agarose purification resin.

Results  The recombinant plasmid pET-30a-Irisin was successfully constructed, and the recombinant expression strain was induced by low temperature and low concentration of IPTG, and the recombinant expression product of high-purity Irisin with a yield of 105 mg/L was obtained by purification with Ni-IDA agarose purification resin.

Conclusion  The optimization of codon and induction can promote the soluble expression of Irisin recombinant protein in Escherichia coli, which lays a solid foundation for the study of its effects and mechanism on different tumor cells.

1.Boström P, Wu J, Jegrychowski MP, et al. A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis[J]. Nature, 2012, 481(7382): 463-468. DOI: 10.1038/nature10777.

2.林幼玉, 王清水, 林尧. FNDC5/Irisin及其与肿瘤关系的研究进展[J]. 福建轻纺, 2023(2): 21-24. [Lin YY, Wang  QS, Lin Y. Progress in the study of FNDC5/Irisin and its relationship with tumors[J]. The Light & Textile Industries of Fujian, 2023(2): 21-24.] DOI: 10.3969/j.issn.1007-550X.2023.02.004.

3.朱英雪. 鸢尾素对上皮性卵巢癌细胞周期调控作用的研究[D]. 青岛: 青岛大学, 2020. [Zhu YX. The effect of Irisin on cell cycle in epithelial ovarian cancer cells[D]. Qingdao: Qingdao University, 2020.] DOI: 10.27262/d.cnki.gqdau.2019.001786.

4.张德国, 谭雪莹, 黄飞, 等. 鸢尾素对胆管癌Hucct-1细胞增殖、凋亡、迁移和侵袭的影响[J]. 中华肝胆外科杂志, 2018, 24(10): 698-702. [Zhang DG, Tan XY, Huang F, et al. Effects of Irisin on proliferation, apoptosis, migration and invasion of human cholangiocarcinoma cell line Hucct-1[J]. Chinese Journal of Hepatobiliary Surgery, 2018, 24(10): 698-702.] DOI: 10.3760/cma.j.issn.1007-8118.2018.10.010.

5.Liu J, Song N, Huang Y, et al. Irisin inhibits pancreatic cancer cell growth via the AMPK-mTOR pathway[J]. Sci Rep, 2018, 8(1): 15247. DOI: 10.1038/s41598-018-33229-w.

6.Zhang D, Tan X, Tang N, et al. Review of research on the role of Irisin in tumors[J]. Onco Targets Ther, 2020, 13: 4423-4430. DOI: 10.2147/OTT.S245178.

7.Vliora M, Nintou E, Karligiotou E, et al. Implication of Irisin in different types of cancer: a systematic review and meta-analysis[J]. Int J Mol Sci, 2022, 23(17): 9971. DOI: 10.3390/ijms23179971.

8.何赛. 人Irisin重组蛋白的表达、分离纯化及活性鉴定[D]. 徐州: 中国矿业大学, 2017. [He S. Expression,  purification and activity identification of human Irisin recombinant protein[D]. Xuzhou: China University of Mining and Technology, 2017.] https://cdmd.cnki.com.cn/Article/CDMD-10290-1016910170.htm.

9.Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3): 209-249. DOI: 10.3322/caac.21660.

10.Zheng RS, Zhang SW, Zeng HM, et al. Cancer incidence and mortality in China, 2016[J]. J Natl Cancer Cent, 2022, 2(1): 1-9. DOI: 10.1016/j.jncc.2022.02.002.

11.Shi G, Tang N, Qiu J, et al. Irisin stimulates cell proliferation and invasion by targeting the PI3K/AKT pathway in human hepatocellular carcinoma[J]. Biochem Biophys Res Commun, 2017, 493(1): 585-591. DOI: 10.1016/j.bbrc.2017.08.148.

12.肖怀腾. FNDC5/Irisin在肝癌中的表达与肝癌侵袭进展的关系及机制的研究[D]. 青岛: 青岛大学, 2022. [Xiao  HT. The relationship between the expression of FNDC5/Irisin in liver cancerand the invasion and progression of liver cancer and its mechanism[D]. Qingdao: Qingdao University, 2022.] DOI: 10.27262/d.cnki.gqdau.2021.001160.

13.Gaggini M, Cabiati M, Del Turco S, et al. Increased FNDC5/Irisin expression in human hepatocellular carcinoma[J]. Peptides, 2017, 88: 62-66. DOI: 10.1016/j.peptides.2016.12.014.

14.殷苏威. 鸢尾素对宫颈癌Hela细胞的生物学作用及机制研究[D]. 青岛: 青岛大学, 2023. [Yin SW. Biological effects and mechanism of irisin on cervical cancer Hela cells[D]. Qingdao: Qingdao University, 2023.] DOI: 10.27262/d.cnki.gqdau.2022.001726.

15.Alshanqiti KH, Alomar SF, Alzoman N, et al. Irisin induces apoptosis in metastatic prostate cancer cells and inhibits tumor growth in vivo[J]. Cancers (Basel), 2023, 15(15): 4000. DOI: 10.3390/cancers15154000.

16.Nowinska K, Jablonska K, Pawelczyk K, et al. Expression of Irisin/FNDC5 in cancer cells and stromal fibroblasts of non-small cell lung cancer[J]. Cancers (Basel), 2019, 11(10): 1538. DOI: 10.3390/cancers11101538.

17.Cebulski K, Piotrowska A, Kmiecik A, et al. The role of Irisin/FNDC5 expression and its serum level in breast cancer[J]. Int J Mol Sci, 2023, 24(10): 8628. DOI: 10.3390/ijms24108628.

18.刘利萍. MTB Hsp16.3重组蛋白构建及通过CX3CR1调控巨噬细胞M2型极化的作用机制研究[D]. 遵义: 遵义医科大学, 2023. [Liu LP. Construction of MTB Hsp16.3 recombinantprotein and its mechanism of regulatingmacrophage M2-type polarization[D]. Zunyi: Zunyi Medical University, 2023.] DOI: 10.27680/d.cnki.gzyyc.2022.000117.

19.Valdez-Cruz NA, Trujillo-Roldán MA. Thermoinducible E. coli for recombinant protein production in inclusion bodies[J]. Methods Mol Biol, 2023, 2617: 17-30. DOI: 10.1007/978-1-0716-2930-7_2.